The long term goal of this project is to engineer imaging probes and techniques to enable biological discovery and biomedical innovation. This proposal aims to develop fluorescent Zn2+ sensors and cell based assays for characterizing Zn2+ levels in different subcellular compartments including secretory granules and cytosol. Zinc ion is the second most abundant transition metal in living organisms and plays numerous important roles in diverse biochemical, biological, and physiological processes. Malfunction of Zn2+ signaling or homeostasis is implicated in a number of human diseases, and there have been increasing interests and efforts to study the regulation and the function of Zn2+ dynamics in living cells. Fluorescent Zn2+ sensors are invaluable tools for such investigations. Despite progress over the past two decades in Zn2+ probe development, there are still pressing needs for engineering new Zn2+ sensors with improved subcellular targeting specificity, dynamic range, and suitable Zn2+ affinity tailored for specific cell organelles. In this proposal, we aim to develop two classes of Zn2+ sensors for imaging Zn2+ activity in the cytosol and in the Zn2+-rich secretory granules; and to combine these sensors with flow cytometry to investigate variations in Zn2+ levels in these two cellular compartments of pancreatic islet cells expressing different isoforms of a granule specific Zn2+ transporter, ZnT8. Human single nucleotide polymorphisms (SNPs) of ZnT8 have been associated with type 2 diabetes (T2D), yet the underlying mechanisms remain elusive, and how ZnT8 affect Zn2+ activity in islet cells needs to be determined. In aim 1, we plan to develop and apply fluorescent Zn2+ sensors to assay Zn2+ activity in secretory granules of pancreatic islet cells including a-cell, b-cell, and d-cell. In aim 2, we plan to develop fluorescent Zn2+ sensors for imaging cytosolic Zn2+ activity by improving the cytosol targeting specificity and Zn2+ binding affinity. In aim 3, we will combine Zn2+ sensors developed in aim 1 and aim 2 with flow cytometry to develop an assay that can simultaneously analyze cytosolic and granular Zn2+ activity in subsets of islet cells. We will apply the assay to compare variations in cytosolic and granular Zn2+ activity in islet cells expressing ZnT8 transporters that control T2D susceptibility. This proposal represents an integral approach by combining sensor design, organic synthesis, flow cytometry analysis, and pancreatic islet biology. We choose pancreatic islets as the biological system for testing, validating, and applying these tools, though probes and techniques developed here should have broad applications in other biological systems.